The authors are grateful to Cristina Godio at Centro Nacional de Biotecnolog&#237;a in Madrid, Spain, Mari Gantner at The Scripps Research Institute, La Jolla, and Anastasia Kralli at Johns Hopkins University, Baltimore, for assistance optimizing this protocol based on the initial work of Kahn et al.18. This work was funded by NIH grants DK114785 and DK121196 to E.S.

This protocol follows all IACUC guidelines of The Scripps Research Institute and the University of Wisconsin &#8211; Madison School of Medicine and Public Health.
1. Collection and digestion of adipose depots (day 1)
<ol>
	<li>Prepare two 1.5 mL tubes for each pup: one for brown adipose tissue (BAT) and one for white adipose tissue (WAT). Add 250 &#181;L of phosphate-buffered saline (PBS) + 200 &#181;L of 2x isolation buffer (123 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), penicillin-streptomycin, and 4% fatty acid-free bovine serum albumin) to each tube. Keep solutions sterile and on ice.</li>
	<li>Place the pups into small chambers (e.g., one well of a 6-well plate), and set them on ice until they become hypothermic. Make sure that there is no direct contact between the pups and the ice. Prick a paw with a tip to assure loss of consciousness, and euthanize the pups by decapitation using sharp scissors. 
	NOTE: if working with different genotypes, prepare an additional tube to collect a tail biopsy (3 mm cut) for genotyping. If genotyping is performed immediately, keep the euthanized pups on ice until the dissection to collect adipose depots.</li>
	<li>Calculate and weigh out the amount of collagenase needed to digest all the depots. Add 50 &#181;L of 15 mg/mL collagenase type I in 2x isolation buffer to each tube. Do not resuspend the collagenase in isolation buffer until all the tissues have been collected.</li>
	<li>To collect subcutaneous WAT, cut the skin around the pup&#39;s abdomen (avoid peritoneal rupture), and gently pull the skin down below the legs. Without taking the skin, carefully collect the fat depot, which will appear as a clear (P1 or younger) or white (P2 and older), thin, elongated tissue attached on the inside or on top of the quadriceps (Figure 1B). Rinse the fat depot in PBS, and place it in one of the tubes containing 250 &#181;L of PBS + 200 &#181;L of 2x isolation buffer. Keep on ice. 
	NOTE: P0 and P1 mice give the best yield. In P0-1 pups, the subcutaneous WAT depot is nearly transparent. In P2 mice and older, the depot is easier to identify because it is already turning white, indicating the presence of fully mature, lipid-loaded, white adipocytes.</li>
	<li>To collect interscapular BAT, pull the skin from the shoulder blades over the head. Lift the BAT - the deep red tissue between the shoulder blades - and carefully make incisions all around it to detach it from the body (Figure 1B). Check for consistency and color; rinse with PBS; place it in the other tube containing 250 &#181;L of PBS + 200 &#181;L of 2x isolation buffer; and keep on ice. 
	NOTE: When harvesting BAT from P2 mice and older, carefully remove the white adipose tissue surrounding the BAT. It consists of a thin, soft, white sheet of adipocytes located between the skin and the BAT, which is deeper between the shoulder blades.</li>
	<li>Once all depots have been collected, gently mince each depot (4-6 times) using small scissors directly inside the tubes.</li>
	<li>Resuspend collagenase type I in the appropriate volume of 2x isolation buffer to obtain a 10x stock solution at 15 mg/mL. Add 50 &#181;L of 10x collagenase to each tube, keeping the tubes on ice until collagenase has been added to all the tubes.</li>
	<li>Invert the tubes quickly to mix, and transfer to a temperature-controlled mixer. Incubate the samples for 30 min at 37 &#176;C on a shaker (1,400 rpm frequency for effective sample mixing).</li>
</ol>
2. Plating preadipocytes (day 1)
<ol>
	<li>After digesting the tissues, place the tubes back on ice. 
	NOTE: From this step onwards, all work is carried out in sterile conditions in a biosafety cabinet.</li>
	<li>Strain the digested tissues through a 100 &#181;m cell strainer into new 50 mL tubes. If working only with WT mice, or if genotypes are known, pool the relevant, dissociated BATs together; repeat this for the WATs. To maximize the cell yield, rinse each tube with 1 mL of isolation medium (Dulbecco&#39;s modified Eagle medium (DMEM) + 20% fetal bovine serum (FBS), 10 mM HEPES, 1% penicillin-streptomycin), and filter it through the cell strainer. If working with unknown genotypes, go to step 2.4 
	NOTE: FBS is an essential determinant of both preadipocyte proliferation and differentiation. Rigorously test different lots of FBS to ensure high performance and consistency between lots.</li>
	<li>Dilute BAT and WAT suspensions to plate 2-3 wells of a 6-well plate for each BAT sample and 4-6 wells of a 6-well plate for each WAT sample. For instance, if 6 BATs and 6 WATs were pooled together, dilute the BAT cell suspension up to 24-36 mL and the WAT cell suspension up to 48-72 mL. Plate 2 mL of the cell suspensions per well.</li>
	<li>For samples with unknown genotypes, keep each sample separate; place a 100 &#181;m cell strainer on top of each well of a 6-well plate, and filter one sample per well. Rinse the tube with 1.5 mL of isolation medium, and pass it through the strainer, making up the final volume to 2 mL per well. Discard the cell strainer, place the lid on the plate, and transfer the plate to the incubator. 
	NOTE: The medium in the wells should look turbid&#160;due to floating blood cells, cell debris, and lipids from lysed cells.</li>
	<li>1-1.5 h after plating, aspirate media&#160;and wash with 2 mL of DMEM without serum. Gently agitate the plate to detach blood cells from the bottom of the wells. After 3 washes, add 2 mL of fresh isolation medium, and transfer the cells to the incubator (37 &#176;C, 5% CO2). 
	NOTE: Check the cells under the microscope. Floating material (blood cells, cellular debris) should be minimal. Brown preadipocytes will appear as small non-translucent cells, whereas white preadipocytes will display a more elongated form. Both brown and white preadipocytes should be tightly attached to the plate.</li>
</ol>
3. Expansion of preadipocyte culture (day 2 to day 5)
<ol>
	<li>On the next day, aspirate the medium, wash the cells with 2 mL of DMEM without serum, and add back 2 mL of the isolation medium.</li>
	<li>Repeat step 3.1 once every 2 days until the cells reach 80-90% confluence. 
	NOTE: For primary brown preadipocytes, reaching 80-90% confluence can take 4-5 days. For primary white preadipocytes, it usually takes 2-3 days. Once the preadipocytes reach 60% confluence, they can be efficiently infected with viral particles for knockdown or overexpression experiments. Infect preadipocytes with an appropriate viral load for up to 8 h. The use of cationic polymers to increase infection efficiency is discouraged, as it often results in toxicity and in a significant reduction of the adipogenic potential of infected preadipocytes.</li>
	<li>To split the cells, coat new destination plates using a sterile solution of 0.1% w/v gelatin (dissolved in distilled water; do not use any heat). Use enough volume to cover the bottom of the well/dish. Incubate plates at 37 &#176;C until the cells are ready to be seeded (at least 10 min). 
	NOTE: This step is optional. Coating of cell culture plates does not affect the yield or differentiation potential, but it substantially simplifies the maintenance and handling of differentiating adipocytes.</li>
	<li>When cells reach sub-confluent density (85-95%), aspirate the medium, wash with PBS, and add trypsin for 3 min to detach the cells. Block trypsin activity by adding 2.5x trypsin volume of isolation medium. Pipette the cell suspension up and down to maximize cell recovery, and transfer into a new tube. Wash the wells with 1 mL of isolation medium, and add it to the first collection.</li>
	<li>Centrifuge the cells for 5 min at 800-1200 &#215; g, aspirate the supernatant, and resuspend the cells in 3-5 mL of the isolation medium. Count the cells, and dilute them to the desired final density. 
	NOTE: For instance, 50,000-80,000 cells/mL (2.5, 1, and 0.5 mL for 6-, 12- and 24-well plates, respectively) will result in fully confluent wells within 72-96 h.</li>
	<li>Aspirate the coating solution in step 3.3, and wash off excess gelatin with PBS. Seed the cells, and return the plates to the incubator until fully confluent.</li>
</ol>
4. Differentiation of white and brown preadipocytes (days 7 - 12)
<ol>
	<li>When preadipocytes become confluent, aspirate the medium, and replace it with differentiation medium consisting of 10% FBS in DMEM containing 170 nM insulin, 1 &#181;M dexamethasone, and 0.5 mM 3-isobuthyl-1-methylxantine. If differentiating BAT preadipocytes, also add 1 nM triiodothyronine (T3). Mark the day on which adipogenic differentiation is induced as day 0 of differentiation. 
	NOTE: Both white and brown preadipocytes can spontaneously differentiate upon reaching confluence. However, to maximize adipocyte differentiation, the traditional pro-adipogenic cocktail described above (plus T3 for BAT preadipocytes) should be used.</li>
	<li>After 48 h (day 2 of differentiation), refresh the medium with maintenance medium consisting of 10% FBS in DMEM and 170 nM insulin (plus 1 nM T3 for BAT). 
	NOTE: On day 2, small lipid droplets accumulating in the cells under the microscope can be observed using standard bright field.</li>
	<li>Repeat step 4.2 once every 2 days until cells are used for experiments. 
	NOTE: On day 4 or 5 of differentiation, the majority of cells will appear loaded with lipids. Terminally differentiating adipocytes can be kept in culture longer or used at this stage for experiments.</li>
</ol>
5. Re-plating for bioenergetics experiments
NOTE: If the intention is to perform bioenergetics studies in mature adipocytes in the 96-well format, the following steps need to be taken. Ideally, cells that have just fully differentiated (day 4 or 5) should be used. The procedure described below starts from 1 well of a 6-well plate.
<ol>
	<li>Prior to trypsin digestion, prepare coating for 96-well plates. Add 50 &#181;L of 0.1% gelatin to each well. Leave the plate in a tissue culture incubator until the cells are ready to be seeded.</li>
	<li>On day 4 or 5 of differentiation, aspirate the medium, wash with 1 mL of PBS, and add 300 &#181;L of 0.25% trypsin-ethylenediamine tetraacetic acid (for 1 well of a 6-well plate). Gently tilt the plate to ensure trypsin completely covers the bottom of the well; incubate for 2 min at room temperature. Block the action of trypsin with 700 &#181;L of maintenance medium, pipette up and down to maximize cell recovery, and transfer the cell suspension into a new 1.5 mL tube.</li>
	<li>Centrifuge the cells at 1200 &#215; g for 5 min. Remove the supernatant, resuspend the pellet in 1 mL of maintenance medium, and count the cells. 
	NOTE: One well of a 6-well plate should yield approximately 1.5-1.8 million cells.</li>
	<li>Dilute the cells to have a final concentration of 60,000 to 80,000 cells/mL for brown adipocytes and 100,000 to 120,000 cells/mL for white adipocytes. Plate 100 &#181;L of the cell suspension per well in gelatin-coated 96-well plates. 
	NOTE: One well of a 6-well plate provides enough cells for up to two 96-well plates. For bioenergetics experiments, low-density plating (30-40% confluence) is needed to enable the accurate measurement of oxygen consumption and avoid oxygen depletion in the wells during the assay.</li>
	<li>Allow the cells to adhere to the bottom of the plate for at least 48 h. If cells are kept in the plate for more than 48 h, refresh the medium after 2 days.</li>
	<li>On the day of the assay, aspirate the medium, and replace it with assay medium. Incubate for 1 h at 37 &#176;C in a non-CO2-controlled incubator, and perform the assay as per the manufacturer&#39;s instructions.</li>
</ol>

<ol><li>Rosen, E. D., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+we+talk+about+when+we+talk+about+fat%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">What we talk about when we talk about fat.</a> Cell. 156 (1-2), 20-44 (2014).</li>
<li>McGarry, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Banting+lecture+2001%3A+Dysregulation+of+fatty+acid+metabolism+in+the+etiology+of+type+2+diabetes%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Banting lecture 2001: Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes.</a> Diabetes. 51 (1), 7-18 (2002).</li>
<li>Kusminski, C. M., Bickel, P. E., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+adipose+tissue+in+the+treatment+of+obesity-associated+diabetes%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">Targeting adipose tissue in the treatment of obesity-associated diabetes.</a> Nature Reviews Drug Discovery. 15 (9), 639-660 (2016).</li>
<li>Waki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+small+molecule+harmine+is+an+antidiabetic+cell-type-specific+regulator+of+PPARgamma+expression%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">The small molecule harmine is an antidiabetic cell-type-specific regulator of PPARgamma expression.</a> Cell Metabolism. 5 (5), 357-370 (2007).</li>
<li>Dominguez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+phenotypic+and+activity-based+profiling+links+Ces3+to+obesity+and+diabetes%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes.</a> Nature Chemical Biology. 10 (2), 113-121 (2014).</li>
<li>Galmozzi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ThermoMouse%3A+an+in+vivo+model+to+identify+modulators+of+UCP1+expression+in+brown+adipose+tissue%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">ThermoMouse: an in vivo model to identify modulators of UCP1 expression in brown adipose tissue.</a> Cell Reports. 9 (5), 1584-1593 (2014).</li>
<li>Ye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRPV4+is+a+regulator+of+adipose+oxidative+metabolism%2C+inflammation%2C+and+energy+homeostasis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">TRPV4 is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis.</a> Cell. 151 (1), 96-110 (2012).</li>
<li>Lynes, M. D., Tseng, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deciphering+adipose+tissue+heterogeneity%5BTitle%5D)+AND+%22Annals+of+the+New+York+Academy+of+Sciences%22%5BJournal%5D)">Deciphering adipose tissue heterogeneity.</a> Annals of the New York Academy of Sciences. 1411 (1), 5-20 (2018).</li>
<li>Schoettl, T., Fischer, I. P., Ussar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneity+of+adipose+tissue+in+development+and+metabolic+function%5BTitle%5D)+AND+%22Journal+of+Experimental+Biology%22%5BJournal%5D)">Heterogeneity of adipose tissue in development and metabolic function.</a> Journal of Experimental Biology. 221 (PT Suppl 1), jeb162958(2018).</li>
<li>Sponton, C. H., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multifaceted+roles+of+beige+fat+in+energy+homeostasis+beyond+UCP1%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Multifaceted roles of beige fat in energy homeostasis beyond UCP1.</a> Endocrinology. 159 (7), 2545-2553 (2018).</li>
<li>Gao, W., Kong, X., Yang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+primary+culture%2C+and+differentiation+of+preadipocytes+from+mouse+brown+adipose+tissue%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Isolation, primary culture, and differentiation of preadipocytes from mouse brown adipose tissue.</a> Methods in Molecular Biology. 1566, 3-8 (2017).</li>
<li>Yu, H., Emont, M., Jun, H., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+differentiation+of+murine+primary+brown%2Fbeige+preadipocytes%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Isolation and differentiation of murine primary brown/beige preadipocytes.</a> Methods in Molecular Biology. (1773), 273-282 (2018).</li>
<li>Church, C. D., Berry, R., Rodeheffer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+study+of+adipocyte+precursors%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Isolation and study of adipocyte precursors.</a> Methods in Enzymology. (537), 31-46 (2014).</li>
<li>Hausman, D. B., Park, H. J., Hausman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+preadipocytes+from+rodent+white+adipose+tissue%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Isolation and culture of preadipocytes from rodent white adipose tissue.</a> Methods in Molecular Biology. (456), 201-219 (2008).</li>
<li>Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H., Frisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retrospective+birth+dating+of+cells+in+humans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Retrospective birth dating of cells in humans.</a> Cell. 122 (1), 133-143 (2005).</li>
<li>Spalding, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+of+fat+cell+turnover+in+humans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Dynamics of fat cell turnover in humans.</a> Nature. 453 (7196), 783-787 (2008).</li>
<li>Wang, Q. A., Tao, C., Gupta, R. K., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tracking+adipogenesis+during+white+adipose+tissue+development%2C+expansion+and+regeneration%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Tracking adipogenesis during white adipose tissue development, expansion and regeneration.</a> Nature Medicine. 19 (10), 1338-1344 (2013).</li>
<li>Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+primary+culture+of+brown+fat+preadipocytes%5BTitle%5D)+AND+%22Sprotocols%22%5BJournal%5D)">Isolation and primary culture of brown fat preadipocytes.</a> Sprotocols. , (2014).</li>
<li>Mills, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accumulation+of+succinate+controls+activation+of+adipose+tissue+thermogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Accumulation of succinate controls activation of adipose tissue thermogenesis.</a> Nature. 560 (7716), 102-106 (2018).</li>
<li>Kajimura, S., Spiegelman, B. M., Seale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brown+and+beige+fat%3A+physiological+roles+beyond+heat+generation%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">Brown and beige fat: physiological roles beyond heat generation.</a> Cell Metabolism. 22 (4), 546-559 (2015).</li>
<li>Pydi, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipocyte+%CE%B2-arrestin-2+is+essential+for+maintaining+whole+body+glucose+and+energy+homeostasis%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Adipocyte β-arrestin-2 is essential for maintaining whole body glucose and energy homeostasis.</a> Nature Communications. 10 (1), 2936(2019).</li>
<li>Sun, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((snRNA-seq+reveals+a+subpopulation+of+adipocytes+that+regulates+thermogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">snRNA-seq reveals a subpopulation of adipocytes that regulates thermogenesis.</a> Nature. 587 (7832), 98-102 (2020).</li>
<li>Galmozzi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PGRMC2+is+an+intracellular+haem+chaperone+critical+for+adipocyte+function%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">PGRMC2 is an intracellular haem chaperone critical for adipocyte function.</a> Nature. 576 (7785), 138-142 (2019).</li>
<li>Brown, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogen-related+receptors+mediate+the+adaptive+response+of+brown+adipose+tissue+to+adrenergic+stimulation%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">Estrogen-related receptors mediate the adaptive response of brown adipose tissue to adrenergic stimulation.</a> iScience. 2, 221-237 (2018).</li>
<li>Shinoda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+and+functional+characterization+of+clonally+derived+adult+human+brown+adipocytes%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Genetic and functional characterization of clonally derived adult human brown adipocytes.</a> Nature Medicine. 21 (4), 389-394 (2015).</li>
<li>Wang, Q. A., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+AdipoChaser+mouse%3A+A+model+tracking+adipogenesis+in+vivo%5BTitle%5D)+AND+%22Adipocyte%22%5BJournal%5D)">The AdipoChaser mouse: A model tracking adipogenesis in vivo.</a> Adipocyte. 3 (2), 146-150 (2014).</li>
</ol>

The authors have nothing to disclose.

Adipose tissue is critical for systemic insulin sensitivity and glucose homeostasis20. Obesity-linked adipocyte dysfunction is tightly associated with the onset of type 2 diabetes. Therefore, greater understanding of the basic biology and physiology of adipose tissue may enable the design of new treatments for metabolic disorders. As a complement to direct functional and transcriptional analysis of mature adipocytes isolated from fat depots21,22...

Obesity results from a chronic imbalance between energy intake and energy expenditure. As obesity develops, white adipocytes undergo a massive expansion in cell size that results in hypoxia in the microenvironment, cell death, inflammation, and insulin resistance1. Dysfunctional, hypertrophied adipocytes cannot properly store excess lipids, which accumulate instead in other tissues where they dampen insulin action2,3. Agents that improve adipocyte function and restore normal lipid partitioning amongst tissues are predicted to be beneficial for the treatment of obesity-associated conditions characterized by insulin resistance such as type 2 diabetes. Phenotypic screens in adipocytes using immortalized cell lines, such as 3T3-L1, F442A, and 10T &#189;, have proven useful to identify genetic factors that regulate adipogenesis and to isolate pro-adipogenic molecules with anti-diabetic properties4,5,6,7. These cell lines, however, do not fully reflect the heterogeneity of cell types present in adipose depots, which includes white, brown, beige, and other adipocyte subtypes with unique characteristics, all of which contribute to systemic homeostasis8,9,10. Further, cultured cell lines often show a diminished response to external stimuli.
In contrast, cultures of primary adipocytes recapitulate more accurately the complexity of in vivo adipogenesis, and primary adipocytes show robust functional responses. Primary preadipocytes are typically isolated from the stromal vascular fraction of adipose depots of adult mice11,12,13,14. However, because the adipose depots of adult animals consist primarily of fully mature adipocytes that have a very slow turnover rate15,16,17, this approach yields a limited quantity of preadipocytes with a low proliferation rate. Therefore, isolation of preadipocytes from newborn mice is preferable to obtain large quantities of rapidly growing cells that can be differentiated in vitro. Here, a protocol has been described, inspired by the initial work with primary brown adipocytes of Kahn et al.18 to efficiently isolate both white and brown preadipocytes that can be expanded and differentiated in vitro into fully functional primary adipocytes (Figure 1A). The advantage of isolating primary cells from newborn, as opposed to adult mice, is that the adipose depots are rapidly growing and are thus a rich source of actively proliferating preadipocytes17. Cells isolated using this protocol have high proliferative capacity, enabling rapid scale-up of cultures. In addition, preadipocytes from newborn pups display higher differentiation potential than adult progenitors, which reduces well-to-well variability in the extent of differentiation and thus increases reproducibility.

<table><tbody><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma-Aldrich</td><td>I7018</td><td></td></tr><tr><td>6-well plates</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>AdipoRed (Nile Red)</td><td>Lonza</td><td>PT-7009</td><td></td></tr><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>BenchMark Fetal Bovine Serum</td><td>Gemini Bioproducts LLC</td><td>100-106</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Cell strainer</td><td>Fisher Scientific</td><td>22363549</td><td></td></tr><tr><td>Collagenase, Type 1&amp;nbsp;&amp;nbsp;</td><td>Worthington Biochemical Corp</td><td>LS004196</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>6442</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5030</td><td>For Bioenergetics studies</td></tr><tr><td>DMEM, High Glucose, Glutamax</td><td>Gibco</td><td>10569010</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Fatty Acid-Free BSA</td><td>Sigma-Aldrich</td><td>A8806</td><td></td></tr><tr><td>FCCP</td><td>Sigma-Aldrich</td><td>C2920</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H1399</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Norepinephrine</td><td>Cayman Chemical</td><td>16673</td><td></td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>75351</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Rosiglitazone</td><td>Sigma-Aldrich</td><td>R2408</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>557368</td><td></td></tr><tr><td>Seahorse XFe96 FluxPak</td><td>Agilent Technologies</td><td>102416-100</td><td>For Bioenergetics studies</td></tr><tr><td>Surgical forceps</td><td>ROBOZ Surgical Instrument Co</td><td>RS-5158</td><td></td></tr><tr><td>Surgical Scissors</td><td>ROBOZ Surgical Instrument Co</td><td>RS-5880</td><td></td></tr><tr><td>ThermoMixer</td><td>Eppendorf</td><td>T1317</td><td></td></tr><tr><td>triiodothyronine (T3)</td><td>Sigma-Aldrich</td><td>642511</td><td></td></tr></tbody></table>

isolation and differentiation of primary white and brown preadipocytes from newborn mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

Section 1 of the protocol will yield a heterogeneous suspension of cells that are visible under a standard light microscope. Filtering of digested tissues with a cell strainer (section 2) will remove undigested tissue. However, some cellular debris, blood cells, and mature adipocytes will pass through (Figure 1C). Gentle washes 1 h after plating will remove non-relevant cells as preadipocytes attach rapidly to the bottom of the well (Figu...

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Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

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Research

JoVE Journal

Biology

11.4K Views.  The Scripps Research Institute, La Jolla, CA, USA. Dit protocol biedt een eenvoudige strategie voor het genereren van celculturele modellen van witte en bruine adipocyten die in vitro getrouw de fysiologie van het vetweefsel dat we in vivo hebben, samenvatten. Dit protocol maakt gelijktijdige isolatie van zowel witte als bruine preadipocyten van pasgeboren muizen mogelijk en de cellen die we met deze methode isoleren, beoordelen een hoge proliferatieve capaciteit en differentiatiepotentieel. Cellen die met deze aanpak worden geïsoleerd, weer...

Video: Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Genetics

Isolation of Preadipocytes from Broiler Chick Embryos

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

Developmental Biology

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Immunology and Infection

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol&#160;reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

This study describes a new method of isolating murine brown adipocytes for gene and protein expression analysis.

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Culturing Murine Adipocytes and Explants for Functional and Imaging Studies

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte&#160;progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

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